Methanolic Extract of Jasminum humile L. Leaves Possess Anxiolytic Activity Mediated via the GABAergic System in the Central Nervous System of Mice

Abstract

Background: 

Jasminum humile L. is one of the traditional medicinal plants from Oleaceae family. It is abundantly available in Afghanistan, Pakistan, Nepal, Burma, the Himalayas, and southwest China. The tea of leaves and barks are used conventionally in the treatment of various diseases such as central nervous system disorder, anti-inflammatory, and antiseptic.

Aim: 

The present study was undertaken to investigate the anxiolytic effect of methanolic extract of J. humile L. leaves (MEJHL).

Materials and Methods: 

Anxiolytic activity of J. humile L. leaves extracts were evaluated using Elevated Plus-Maze (EPM) and light/dark exploration models. The motor coordination and sedative effect were also observed using Rota Rod apparatus, actophotometer, and ketamine-induced sleeping time, respectively. Brain neurotransmitters like GABA level were estimated by validated methods.

Results: 

From the acute toxicity studies, extracts of J. humile L. leaves were found to be nontoxic. Among all extracts, the only methanolic extract showed significant (P < 0.05) anxiolytic activity. Preliminary phytochemical screening revealed the presence of carbohydrates, flavonoids, and alkaloids in the MEJHL. The results of anxiolytic models clearly depicted that MEJHL at the dose of 200 mg/kg significantly (P < 0.05) increased the number of entries and the average time spent in the open arms and light compartment of the EPM and light/dark models, respectively. In the case of motor coordination activity, the MEJHL does not cause any significant effect (at any dose) on the latency to fall off from the Rota Rod bar compared to the control group. Moreover, no remarkable effects in actophotometer and on ketamine-induced sleep latency and total sleeping time induced by ketamine was observed. Results of neurotransmitter estimation in the whole brain and the cerebellum revealed the increased concentration of GABA was similar to standard drug diazepam.

Conclusions: 

The presence of alkaloids in the methanolic extract of this plant can serve as a potential resource for natural psychotherapeutic agents against anxiety with less sedation.

Introduction

Anxiety is a psychological and physiological state characterized by cognitive, somatic, emotional, and behavioral components which combine together to create an unpleasant feeling that is typically associated with uneasiness, apprehension, fear, or worry. There is a persistent and disproportionate fear unrelated to any genuine risk.[¹] At present, anxiety is the most frequent psychiatric condition commonly found with complex causes and etiology. According to epidemiological surveys, one-third of the population is affected by this disorder during their lifetime, being more common in women as compared to men. Majority of the population suffers from this psychiatric illness due to unhealthy lifestyles, physical inactivity, and junk diet. Adherence to lifestyle recommendations for the prevention of anxiety remains a critical issue. When the symptoms of anxiety become intolerable and if counseling is not sufficient, drug treatment can be considered as a means of helping patients to cope up with their anxiety.[²] Till date, the efficacy of the drugs for treating these conditions is very limited so the need for newer, better-tolerated, and more effective treatments still remains high. The most widely prescribed medications for anxiety disorders belong to the category of benzodiazepines. However, their clinical uses are limited due to their severe adverse effects such as psychomotor impairment, potentiating of other central depressant drugs, and dependence liability.[³] Therefore, herbal therapies should be considered as alternative/complementary medicines with the additional benefit of being safe and better tolerated. Recently, the search for novel pharmacotherapy from medicinal plants for psychiatric illnesses has progressed significantly.[⁴] This has been reflected in the large number of herbal medicines whose psychotherapeutic potential has been assessed in a variety of animal models.

Jasminum humile L. (Oleaceae) commonly known as Peeli chameli and Pitmalti, originating in parts of tropical India and Burma and is widely known for its perfumery. According to the data available in the traditional literature, i.e. Indian Materia Medica it is used in treating whooping cough, asthma, as a tonic for the heart and bowels, intestinal problems, and ringworm infections. The activity of this plant has been described as antispasmodic, antidepressant, central nervous system (CNS) disorder, anti-inflammatory, antiseptic, aphrodisiac, expectorant, and tonic (uterine) effects. Its leaves are used to treat skin disorders which resemble the use of modern topical anti-inflammatory drugs. The milky juice of the plant is used for destroying the unhealthy lining walls of chronic sinuses and fistulas.[^5,6] The plant is a rich source of important constituents such as indole alkaloids, iridoids, coumarins, and tannins. Alpha-amyrin, betulin, friedelin, lupeol, betulinic, oleanolic, ursolic acid, beta-sitosterol, and secoiridoid glycoside are found abundantly in the leaves.[^7-9] Despite its remarkable uses, detailed data related to its biological properties is still lacking_. Till today, no significant work has been conducted to validate the traditional uses of J. humile L., so the present study has been made to establish the scientific validity for the antianxiety property of J. humile L., that contribute for beneficial uses of this plant in the management of anxiety disorder.

Materials and Methods

Drugs and chemicals

Diazepam (Sigma-D0899) and Ketamine hydrochloride (Sigma-K2753) were purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals were of analytical grade and commercially procured from S.D. Fine Chem. Ltd. (Mumbai, India).

Plant material

The leaves of J. humile L. were procured and identified from a cultivated source from Herbal Nature Park, Chuharpur, forest Division (Yamunanagar) and authenticated from NISCAIR (Delhi) with Reference number NISCAIR/RHMD/Consult/2010-11/1679/277.

Preparation of extracts

Leaves of J. humile L. were dried in shade and powdered. The powdered leaves (500 g) were subjected to successive soxhlet extraction by using different solvents in increasing order of polarity, i.e. petroleum ether (60°C–80°C), chloroform, methanol, and water. The powdered drug material was dried in hot air-oven below 50°C before extraction. Each extract was concentrated by distilling off the solvent and then evaporating to dryness on the water bath. All the extracts were weighed and also the percentage of the yield was calculated in terms of the air-dried weight of the plant material. The extracts were stored for future animal studies.

Phytochemical screening

The dried extracts of J. humile L. leaves were dissolved in their respective solvents and were screened for the presence of different classes of secondary metabolites such as alkaloids, flavonoids, carbohydrate, steroids terpenes, polyphenols, and tannins.

Test animals

The experimental animals (Swiss albino mice [20–25 g] of either sex] were procured from the Institutional Animal House. The animals were maintained in a well-ventilated room with 12:12 h light/dark cycle in cages. Standard pellet feed and drinking water ad libitum were provided. Animals were acclimatized to laboratory conditions at least 1 week before the initiation of experiments. The animal care and experimental protocols were in accordance with Institutional Animal Ethical Committee. All the procedures and protocols used for experiments were prior approved by the Institutional Animal Ethics Committee (MMCP/IEC-0436/10/012) and conducted according to the Indian National Science Academy Guidelines for the use and care of experimental animals.[¹⁰]

Acute toxicity studies

The acute oral toxicity study was carried out as per the guidelines set by Organization for Economic Co-operation and Development received from the Committee for the Purpose of Control and Supervision of Experiments on Animals. One-tenth of the median lethal dose (LD₅₀) was taken as an effective dose.[¹¹]

Treatment

Animals were divided into 14 (I-XVIII) groups. Group I was control and was given vehicle, consisting of Tween 80, in a dose of 1 ml. Group II was a positive control and was given standard drug, diazepam (2 mg/kg) suspended in the vehicle. Group III-XVIII were treated as test groups and were given petroleum ether, chloroform, methanol, and water extracts of J. humile L. leaves at different doses, namely, 50, 100, 200, and 400 mg/kg, respectively. All the test solutions, standard drug, and vehicle were administered orally 45 min before elevate antianxiety activity.

Model used for anxiety activity

Light/dark exploration test

The apparatus consisted of two acrylic boxes. Two distinct chambers, a black chamber (20 cm × 30 cm × 30 cm) painted black and other open chamber made up of transparent acrylic (30 cm × 30 cm × 30 cm). The two chambers were connected through a small open doorway (8 cm × 8 cm) situated on the floor level at the center of the partition.[¹²] One box was made dark by covering its top with plywood and a 10 W lamp illuminated the other box. The light source was placed 25 cm above the open box. The mice were placed individually in the center of the lit box and observed for the next 5 min for the time spent in lit and dark boxes. Each mice was placed individually in the light compartment and observed for the next 5 min for the numbers of the crossing between two compartments and time spend in the light and dark compartment.[¹³]

Elevated Plus Maze Model

The Elevated Plus Maze (EPM) model of anxiety has been extensively used for the evaluation of novel anxiolytic agents and to investigate the psychological and neurochemical basis of anxiety. It consists of two open arms (16 cm × 5 cm for mice and 50 cm × 10 cm for rats), two closed arms (16 cm × 5 cm × 12 cm for mice and 50 cm × 10 cm × 40 cm for rats), and an open roof with the entire maze elevated (25 cm for mice and 50 cm for rats) from the floor.[¹⁴] The animals were placed individually in the center of the maze, with their head facing towards open arms. The stopwatch was started and the following parameters were noted critically for the period of 5 min.

1. First preference of mice to open and closed arm
2. Number of entries in open/closed arms (an arm entry defined as the entry of four paws into the arm)
3. Average time of each animal spends in each arm (average time = total duration in the arm/number of entries).

Rota Rod apparatus (motor coordinator test)

Rota Rod apparatus is commonly utilized for the assessment of neuromuscular coordination in rodents.[¹⁵] Motor coordination tests can be performed by comparing the latency to fall between the different groups. Mice were trained for 3 days before the test to remain on the rotating bar (speed 20 rpm) for at least 3 min with three trials per day. On the test day, mice were arbitrarily divided into six groups. Normal saline (vehicle-treated control group), extracts (50, 100, 200, and 400 mg/kg, p.o.), and diazepam (3 mg/kg, p.o) were administrated orally to them. Forty minutes after that administration, mice were put on the rotating bar and the latency until fall or their ability to remain itself on the rotating bar during a 5 m session was recorded at 0, 0.5, 1, 1.5, and 2 h.[¹⁶]

Evaluation of sedative activity

Locomotor activity

The locomotor activity was measured by using an actophotometer. The movement of the animal interrupts a beam of light falling on a photocell, by which a count was recorded and displayed digitally. Different doses (50, 100, 200, and 400 mg/kg) of J. humile L leaves extract and vehicle were administered for 5 days once daily p.o. and the last dose was given on the 7^th day, 60 min before the start of the experiment. The standard drug was given at a dose of 2 mg/kg p.o. 60 min before initiating the experiment and the animals were kept in the actophotometer individually. The locomotor activity was measured for a time period of 5 min.[¹⁷]

Ketamine-induced sleeping time

The effect of J. humile L. leaves extracts on ketamine-induced sleeping time was estimated.[¹⁸] Experimental mice were randomly divided into control (group I) and treatment groups (Group II, III, IV, V, and VI) containing six animals in each group. Group I received the vehicle and Groups III, IV, V, and VI received J. humile L. leaves extracts (50, 100, 200, and 400 mg/kg, respectively), 1 h before the intraperitoneal administration of ketamine (100 mg/kg). Group II, mice were treated with diazepam (2 mg/kg, p.o.) 30 m before ketamine injection. The time duration between the ketamine administration until the loss of the animal righting reflex was noticed as the onset of sleep, while the time from the loss to regaining of the righting reflex as the time duration of sleep.[¹⁹]

Neurotransmitters estimation (brain GABA estimation)

The concentration of GABA in the whole brain was estimated by using the established method.[²⁰] Brains of mice were quickly harvested after finishing behavioral model trails, and isolated brains were weighed and transferred to 5 ml of ice-cold trichloroacetic acid (10% w/v). Brains were homogenized and centrifuged at 10,000 g for 10 m at 0°C. In 0.1 ml of tissue extract, added 0.2 ml of ninhydrin solution (0.15 M) in a 0.5 M carbonate bicarbonate buffer (pH 9.9), which was incubated in a water bath at 60°C for 30 m and after cooling it was treated with 5 ml of copper tartrate reagent (0.03% tartaric acid, 0.03% copper sulfate and 0.16% disodium carbonate). A fluorescence reading was taken at wavelengths of 377- 451 nm in a spectrofluorometer after 10 m.

Statistical analysis

Results were expressed as mean ± standard error of the mean differences among data were determined using one-way ANOVA followed by Dunnett’s t-test (GraphPad Software, Inc, California (US) Prism software for Windows 6.07). Differences between the data were considered significant at P < 0.05.

Results

Phytochemistry

The results of phytochemical screening clearly indicated the presence of carbohydrates, alkaloids, and flavonoids in methanolic extract of J. humile L. [Table 1].

Table 1: Phytochemical screening of methanolic leaves extract of Jasminum humile L.

Acute toxicity studies and dose selection

From the acute toxicity study, MEJHL did not produce any mortality orally up to 2 g/kg, but mice manifested signs of sedation such as quiescence and reduced locomotion at the higher dose (more than 1000 mg/kg). No visible signs of delayed toxicity and mortality were observed when the animals were monitored for 7 days. Hence, based on toxicity data the starting test dose for this activity was taken as 50 mg/kg body weight for the extracts.

Light and dark model

The vehicle-treated mice spent time (79.8 ± 8.4 s) and the number of entries (7.3 ± 0.9) in the light compartment of the LTD model. Diazepam-treated mice significantly (P < 0.05) increased the time spent (167.3 ± 7.2 s) as well as the number of entries (16.6 ± 1.5) in the light compartment as compared with vehicle-treated control group. Among the various extracts of Jasminum humile L. leaves, only methanolic extract of Jasminum humile L. leaves (MEJHL) significantly (P < 0.05) increased the mean number of entries and time spent by mice in the light compartment of light-dark apparatus at the dose of 200 mg/kg with respect to control (vehicle) group. That shows MEJHL produced anxiolytic activity in light and dark experiments on mice [Table 2 and Figures 1, 2].

Table 2: Effect of various extracts of leaves of Jasminum humile L. on behavior of mice using light and dark exploration test

Figure 1: Effect of methanolic extract of Jasminum humile L. leaves on time spent by animal using light and dark model (n = 6), *P < 0.05

Figure 2: Effect of methanolic extract of Jasminum humile L. leaves on number of entries in light area by animal using light and dark model (n = 6), *P < 0.05

Elevated Plus Maze Model

The anxiolytic behavior of mice on the EPM model was confirmed by diazepam. The results showed an average time spent by the animals in open arms increased from 27.74 ± 6.3 (s) in vehicle-treated control group to 121.24 ± 5.3 (s) and 120.12 ± 6.7 (s) in methanolic extract-treated group at a dose of 200 and 400 mg/kg respectively among it all other extracts of Jasminum humile L. leaves. This indicates that MEJHL showed significant (P < 0.05) antianxiety activity as compared to the control (vehicle) group [Table 3 and Figures 3, 4].

Table 3: Effect of various extracts of leaves of Jasminum humile on behavior of mice using elevated plus-maze model

Figure 3: Effect of methanolic extract of Jasminum humile L. leaves on time spent by animal using Elevated Plus-Maze model (n = 6), *P < 0.05

Figure 4: Effect of methanolic extract of Jasminum humile L. leaves on number of entries in open arm by animal using Elevated Plus-Maze model (n = 6), *P < 0.05

Rota Rod apparatus

In this test, the standard drug (diazepam) significantly (P < 0.05) decreased the latency (79.5 ± 6.7 after 2 h treatment) to fall off from the rotating rod when compared to the vehicle control (263.4 ± 9.4 after 2 h treatment). However, MEJHL at doses 50, 100, 200, and 400 mg/kg did not produce any significant (P < 0.05) reduction in the time spent by the mice on the revolving rod when compared to the control. The result from the Rota Rod apparatus test showed that MEJHL did not produce muscle relaxation action at selected doses [Table 4].

Table 4: Effect of methanolic extract of Jasminum humile L. leaves on Rota Rod apparatus

Locomotor activity

Locomotors activity is considered as an index of alertness and a decrease in its value indicates a considerable sedative effect. A significant (P < 0.05) decrease in the locomotor score was observed with diazepam (2 mg/kg) when compared to the control animals but all the doses of MEJHL did not show [Figure 5] any decrease in the locomotors score, thus indicating that MEJHL did not possess any sedative effect.

Figure 5: Effect of methanolic extract of Jasminum humile L. leaves on animals in locomotor activity. n = 6, *P < 0.05 (one-way ANOVA followed by Dennett’s test)

Ketamine-induced sleeping time

In vehicle treated control mice, the righting reflex (sleep latency period) was lost after 121 ± 9 s of ketamine injection and total sleep time was 1120 ± 119 s while with diazepam (2 mg/kg) the righting reflex in mice was lost after 67 ± 4 s, with the significant increase in the total sleep time by 2019.8 ± 181 s. MEJHL (30 m prior to ketamine) at doses of 50, 100, 200, and 400 mg/kg did not significantly change the latency to sleep [Figures 6 and 7].

Figure 6: Effect of methanolic extract of Jasminum humile L. leaves on sleeping time (s) (n = 6), *P < 0.05

Figure 7: Effect of methanolic extract of Jasminum humile L. leaves on time latency before sleep (s) (n = 6), *P < 0.05

Estimation of GABA neurotransmitter

Brain GABA content was significantly lower in stressed mice as compared to unstressed mice. MEJHL has significantly (P < 0.05) increased the GABA levels in the whole brain and the cerebellum in stressed mice as compared with their respective control groups. On the other hand, diazepam also significantly (P < 0.05) increased the GABA content in the whole brain (other than cerebellum) and cerebellum alone in stressed mice [Table 5].

Table 5: Effect of methanolic extract of Jasminum humile L. leaves on brain GABA levels

Discussion

Thousands of medicinal plants and plant extracts are used as traditional medicine for the treatment of a wide range of diseases such as rheumatoid arthritis, diabetes, obesity, malaria and not the least for neurological disorders affecting the CNS.[²¹] Despite the widespread traditional use of Jasminum humile L. for treating various anxiety disorders, there are no reports of scientific evaluation of its anxiolytic activity. Based on the traditional claims and the reported activities, the present study was aimed at evaluating the anti-anxiety property of Jasminum humile L. leaves in comparison with control and standard drug using battery of animal models.

The EPM animal model is validated test and widely used for assaying anxiolytic substances such as benzodiazepines through activation of GABA receptors. The fear due to height induces anxiety in the animals when placed on the EPM.[²²] Anxiolytic agents are expected to increase motor activity, which is measured by the time spent by the animal in the open arms. In the current study, amongst various extracts, MEJHL significantly increased mean time spent and mean number of entries by mice in open arms of EPM apparatus at all doses with respect to control, thereby producing anti-anxiety activity. The higher dose (200 mg/kg) exhibits significant anxiolytic effect similar to that produced by standard drug (diazepam). There were no statistical differences between these two groups. The anxiolytic effect was additionally confirmed through two compartmental exploratory models, i.e. light-dark exploration.[²³] It has been accepted that the time spend by mice in the lighted side of the box is the most helpful and consistent parameter of anxiety.[²⁴] In the investigation, MEJHL significantly increased mean time spent and mean number of entries by mice in illuminated side box of light and dark apparatus at dose 200 mg/kg with respect to control, and as also expected by diazepam.

Rota Rod apparatus is widely used to evaluate peripheral neuromuscular blockade and coordination aspects of motor function.[¹⁵] Moreover, modification in motor coordination on the Rota Rod test suggests that the diminished locomotor action exerted through peripheral neuromuscular blockage or centrally mediated impairment of motor function.[²⁵] Our findings showed that MEJHL did not significantly alter the time of performance (motor coordination) on the bar of Rota Rodlike diazepam (1 mg/kg), suggesting that the anxiolytic-like activity without myorelaxant effect.

Locomotor activity is taken into account as an index of alertness and a decline in that indicates a sedative effect.[²⁶] In contrast to diazepam, MEJHL had no effect on locomotor activity counts (actophotometer). In ketamine-induced sleeping time test, diazepam decrease sleep latency and increase sleeping time. This investigation revealed an overactivity of the dopaminergic pathway in response to ketamine administration.[²⁷] Treatment with MEJHL has not shown a statistically changed latency to sleep but the slight increase in sleeping time only at a dose of 400 mg/kg induced by ketamine.

The adrenergic and dopaminergic systems have also been shown to play a role in the management of the mechanisms of anxiety.[²⁸] The etiology of most anxiety disorders is still not fully understood and ambiguous, but various studies have shown the involvement of GABAergic, serotonergic neurotransmission in the etiology, expression, and treatment of anxiety.[²⁹] GABA seems to assume a vital role in the pathogenesis of several neuropsychiatric disorders and the significant number of therapeutic agents used to treat psychiatric illness by enhancing the GABA action on GABA receptors including benzodiazepine.[³⁰] In the present study, the level of GABA content was significantly (P < 0.05) increased in the cerebellum and whole brain (other than cerebellum) by diazepam and MEJHL (200 mg/kg) when compared with the control group which clearly indicates that diazepam and MEJHL express their anxiolytic behavior by GABAergic system.

The level of GABA content in the brain of mice and anxiolytic behavior effect of the leaves extract was more prominent at 200 mg/kg and doses higher or lower than this did not show consistent effects. Hence, the result of this study showed that, MEJHL (200 mg/kg) has significant anxiolytic activity as compared to the control group. Further, it was found that alkaloids have significantly attributed to its effect on CNS and benzodiazepine receptors.[^31-33] Therefore, alkaloids present in MEJHL as investigated by phytochemical screening might be responsible for the anti-anxiety activity without inducing sedation. However, further studies are still required to identify the important phytoconstituents which are present and elucidate the mechanism responsible for the observed anxiolytic effect of methanolic extract at dose 200 mg/kg and to explain the exact anxiolytic mechanism.

Conclusions

From the above observations, we can conclude that MEJHL possesses anxiolytic activity at 200 mg/kg dose level which is comparable with the standard drug diazepam. However, further studies are required to know the exact mechanism of action of MEJHL as an anxiolytics agent.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Acknowledgments

We would like to acknowledge Maharishi Markandeshwar (Deemed to be University) for providing facilities for research.